The importance of protein therapeutics has grown immensely over the past decades, as this class of drugs is targeting therebefore untreatable diseases. The formulation of protein drugs is challenging as their molecular size is very different from traditional low molecular weight substances. Furthermore, proteins have several other characteristics like their secondary and tertiary structure essential for proper functionality. They are susceptible to many types of degradation or modification and their formulation has to ensure stability and has to remain within their efficacious and safe target doses.
Other problems concerning proteins as pharmaceutical compounds are caused within the patient, e.g. immunogenic reactions, rapid clearance from the body leading to a reduced half-life, as well as proteolytic cleavage destructing the drug before performing its action (Brown, L. R., Expert Opinion Drug Deliv. 2 (2005) 29-42).
To address these problems one approach is to include chemical alterations in protein molecules, e.g. covalently attaching one or more poly (ethylene glycol) residues to the protein drug. This procedure is called PEGylation. The advantages of PEGylation are the increase in molecular weight and hydrodynamic size of the resulting conjugate. The new conjugate is less easy accessible for proteolytic enzymes as well as neutralizing antibodies or immune cells. As a result PEGylation is useful to prevent the drug from undesired cleavage and also decreases the possibility of allergic side-effects. The increased size is also influencing the clearance from the body, because the protein-PEG conjugate is too big to be removed by renal ultrafiltration (Veronese, F. M. and Pasut, G., Drug Discovery Today 10 (2005) 1451-1458; Brown, L. R., Expert Opinion Drug Deliv. 2 (2005) 29-42). Poly (ethylene glycol) itself improves the properties of the new drug immensely. In addition PEG's lack of toxicity is a major advantage. Taken together PEGylation is a well known method for increasing the blood circulation life time of a protein pharmaceutical (Kozlowski, A. and Harris, M. J., Journal of Controlled Release 72 (2001) 217-224). A variety of PEGylated protein drugs is commercially available, e.g. PEG-Intron® from Schering Plough, Somavert® from Pfizer and Pegasys® from Roche Pharmaceuticals (Pasut, G., et al., Expert Opinion on Therapeutic Patents 14 (2004) 859-894).
There is great interest for enzymatic site-specific modifications with mild conditions, high chemo- and regio-specifity and proper product yields. Several attempts in this field have been made. Mao et al. (Mao, H., et al., J. Am. Chem. Soc. 126 (2004) 2670-2671) developed a method of sortase-mediated protein ligation for C-terminal modification. Sato (Sato, H., Advanced Drug Delivery Reviews 54 (2002) 487-504) established a system using transglutaminase to introduce poly (ethylene glycol) site-specifically into intact and chimeric proteins inheriting a special substrate sequence for transglutaminase. Immunoglobulin A protease (IgA protease) has been used by Lewinska et al. (Lewinska, M., et al. Bioconjugate Chemistry 15 (2004) 231-234) to modify the N-terminus of native proteins. A mutated trypsin enzyme has been developed by Hoess et al. (WO 2006/015879).